Biochemistry of Melanization in Mosquitoes

Melanization/pigmentation is an important physiological phenomenon in insects,which is involved incuticular tanning,parasite encapsulation,and egg shell hardening.During the past few years,we have beenusing mosquito eggs as a model to study the detailed biochemistry of melanization reactions in mosquitoes.Chorion of newly oviposited eggs is white and soft,but the chorion becomes black and hard within 3～4 hfollowing oviposition. Once the chorion turns black,the eggs become resistant to desiccation and solubiliza-tion.Floodwater mosquitoes,such as Aedes aegypti,oviposit on substrates at the edge of water and theeggs hatch only after being flooded with water following adequate rainfall.Consequently,the ability towithstand the desiccation and other environmental adversities is critical for egg survival.Our research deal-ing with chorion melanization in mosquitoes is aimed at understanding the biochemical processes and mech-

Semi-conductive properties of melanization of chromate zinc plates

The anodic polarization curves of chromate zinc plates were studied under the condition of with and without illumination. It is found that the chromate film of zinc plate is characterized by an n type semi conductive property. The illumination on the surface and the addition of Pb in the surface raises the anodic photocurrent and plays a part of the n type property in the film. Contrarily, Ag in the film plays a part of the p type dopant. The effects of quantitative Pb 2+ and Ag + in bath on the melanization(black patina) of zinc plate after chromate treatment also has been studied by comparing the lightness differences Δ L by means of a whiteness meter. It is found that the lightness decreasing process (the melanization rate) is consistent with the Wagner equation Δ L =kt 1/2 +C, and that Pb in the film increases k , on the other hand, Ag in the film decreases k of the melanizing rate.[

Two Hypotheses of “Melanization” on Electrogalvanized Steel Surface

Melanization is harmful to the lightness of galvanized steel surface and also reduces the bonding force between a paint coat and the steel sheet. Experimental results showed that the hypothesis of Zn-Pb codeposition does not hold true, while the fact that PbSO4 micelles in sulfate bath settle on the coating surface support the second hypothesis. The authors come to the conclusions that (1) Any measure to eliminate PbSO4 micelles before or after passivation of the coating will prevent melanization; (2) PbSO4 micelles adsorbed on the surface of passivated film promote the growth of oxygen-deficient Zn oxide and result in the generation of melanization.

Effects of mosquito hemocytes on normal and degenerated oocysts of Plasmodium yoelii and their role in oocyst melanization

Anopheles stephenst, infected with Plasinodium yoelii, was fed with 10% sucrose solution contatning 1% difluoromethylornithine (DFMO) to induce oocyst degeneration of the malaria parasites. On the 11th day after infection, the effects of mosquito hemocytes On the normal and degenerated oocysts were observed under transmission electron microscope. In the control group, no hemocyte could be found around the normally-developed oocysts. In the DFMO-treated group, all the oocysts underwent degeneration in various degrees and some of them were melanized. All the oocysts were attached by one or more hemocytes of the kind of granulocytes.There were many granules with microtubular structure in the cytoplasm of the hemocytes and in the space between the hemocyte and the oocyst. The findings suggest that the degeneration of oocysts can attract the hemocytes to attach around them and the latter can release granules and possibly other substances to cause encapsulation and melanization of the oocvsts.

A secreted phospholipase A_(2) (BmsPLA_(2)) regulates melanization of immunity through BmDDC in the silkworm Bombyx mori

Insect immune-associated phospholipase A_(2) (PLA_(2)) is an important target of pathogen invasion. Melanization, an effective defense response, has significant correlations with other immune responses to coordinate immune attack against invaders. However, the effect of PLA_(2) on melanization has not yet been reported in insects or other arthropods. In this work, we cloned a PLA_(2) gene (BmsPLA_(2)), and its protein had characteristic features of secreted PLA_(2) (sPLA_(2)). After injection of bacteria, BmsPLA_(2) expression and sPLA_(2) activity in hemolymph significantly increased. BmsPLA_(2) fluorescence was transferred from the cytoplasm to the cell membranes of circulating hemocytes. These results indicated that BmsPLA_(2) was related to hemolymph immunity in silkworms. Interestingly, reducing BmsPLA_(2) by RNA interference decreased melanosis (melanistic hemocytes) levels in vivo and in vitro, while BmsPLA_(2) overexpression had the opposite effect. The larval survival and melanization rate in the hemocoel both slowed depending on the PLA_(2) inhibitor dosage. These results demonstrated that BmsPLA_(2) plays a role in melanization during the immune process of silkworms. Surprisingly, the level of BmDDC matched the degree of melanization in various observations. BmDDC expression showed a significant increase, with the peak occurring later than that of BmsPLA_(2) after injection of bacteria, implying that BmsPLA_(2) was activated prior to BmDDC. Moreover, the alteration of BmsPLA_(2) by RNA interference or overexpression led to altered BmDDC levels. These results suggested that BmsPLA_(2) regulates the melanization response in silkworms through BmDDC. Our study proposes a new regulatory mechanism of the melanization response and new directions for understanding the complex immune networks of insects.

Melanization in living organisms: a perspective of species evolution

Eumelanin is a heteropolymer that is generally composed of hydroxylated indole residues and plays diverse protective functions in various species.Melanin is derived from the amino acid tyrosine and production of melanin is a highly complex oxidative process with a number of steps that can either proceed enzymatically or non-enzymatically.Although melanin plays important protective roles in many species,during melanization,particularly in steps that can proceed non-enzymatically,many toxic intermediates are produced,including semiquinones,dopaquinone,indole-quinones and moreover,the production of many reactive oxygen species.To mitigate the production of reactive species,a number of proteins that regulate the biochemical process of melanization have evolved in various living species,which is closely related to adaptation and physiological requirements.In this communication,we discuss differences between non-enzymatic and enzymatic processes of melanization and the enzymatic regulation of melanization in difference species with an emphasis on differences between mammals and insects.Comparison between melanization and insect sclerotization is also emphasized which raises some interesting questions about the current models of these pathways.

Insecticide toxicity associated with detoxification enzymes and genes related to transcription of cuticular melanization among color morphs of Asian citrus psyllid

The Asian citrus psyllid (Diaphorina citri Kuwayama) is known to exhibit abdominal color polymorphisms. In the current study, susceptibility to four insecticides was compared among orange/yellow, blue/green and gray/brown color morphs of field collected D. citri. The LD50 values and 95% fiducial limits were quantified for each insecticide and color morph combination and ranged between 0.10 ng/μL (0.06-0.10) and 6.16 ng//μL (3.30-12.50). Second, we measured the detoxification enzyme activity levels of orange/yellow, blue/green and gray/brown color morphs for cytochrome P450, glutathione "S-transferase, and general esterase. The mean P450 activity (equivalent units) was significantly lower in gray/brown (0.152 ± 0.006) and blue/green morphs (0.149 ± 0.005) than in the orange/yellow morphs (0.179 ± 0.008). GST activity (/imol/min/mg protein) was significantly lower in the orange/yellow morph (299.70 ± 1.24) than gray/brown (350.86 ± 1.19) and blue/green (412.25 ±1.37) morphs. The mean EST activity (/xmol/min/mg protein) was significantly higher in blue/green (416.72±5.12) and gray/brown morphs (362.19 ± 4.69) than in the orange/yellow morphs (282.56 ± 2.93). Additionally, we analyzed the relative expression of assortment genes involved in cuticular melanization and basal immunity. The transcripts of Dopa Decarboxylase and Tyrosine Hydroxylase were expressed higher in blue/green and gray/brown than orange/yellow morphs. The transcription results paralleled the susceptibility of D.citri to organophosphate, neonicotinoid and pyrethroid insecticides. GST and EST activity may also be correlated with low levels of insecticide susceptibility. Cuticular melanization could be a factor for the development of resistance to insecticides among different color morphs.

Melanization induced by Heliothis virescens ascovirus 3h promotes viral replication

Melanization is an important innate immune defense mechanism of insects,which can kill invading pathogens.Most pathogens,for their survival and reproduction,inhibit the melanization of the host.Interestingly,our results suggested that after infection with Heliothis virescens ascovirus 3h(HvAV-3h),the speed of melanization in infected Spodoptera exigiia larval hemolymph was accelerated and that the phenoloxidase(PO)activity of hemolymph in larvae infected with HvAV-3h increased significantly(1.20-fold at 96 hpi,1.52-fold at 120 hpi,1.23-fold at 144 hpi,1.12-fold at 168 hpi).The transcription level of the gene encoding S.exigua prophenoloxidase-1(SePPO-1 gene)was upregulated dramatically in the fat body during the middle stage of infection.In addition,when melanization was inhibited or promoted,the replication of HvAV-3h was inhibited or promoted,respectively.In conclusion,infection with HvAV-3h can markedly induce melanization in the middle stage of infection,and melanization is helpful for HvAV-3h viral replication.

Bombyx mori U-shaped regulates the melanization cascade and immune response via binding with the Lozenge protein

Zinc finger protein,an important transcription factor,regulates gene expression associated with various physiological and pathological processes.U-shaped,belong to the Friend of GATA(FOG)transcription factor,plays a crucial role in hematopoiesis by interacting with the GATA transcription factor as a co-factor.However,little is known about its functions in insects.In the present study,a U-shaped cDNA was identified and characterized from the silkworm Bombyx mori and its potential roles in innate immunity investigated.The predicted silkworm U-shaped amino acid sequence contained a classical nuclear localization signal(NLS)motif“GESSPKRRRR”at position 45CU459,and arginine residues at position 456 and 478 are the critical sites of the NLS.U-shaped mRNA was detected in all tested tissues of the B.mori;however,the highest levels were found in the hemocytes.U-shaped mRNA expression levels were upregulated in the hemocyte after the Escherichia coli and Staphylococcus aureus challenge.Furthermore,U-shaped knockdown significantly reduced the melanization process and suppressed the expression of melanization-associated genes,including PPO1,PPO2,PPAE and BAEE.In addition,U-shaped interacts with Lozenge protein to regulate the innate immune response of the insect.Our results revealed that U-shaped binds directly to Lozenge protein to modulate the melanization process and innate immune responses in silkworm.

芦荟素对Melan-a鼠黑素细胞株黑素生成及其相关基因表达的影响

目的:研究芦荟素对Melan-a鼠黑素细胞株的黑素生成及其相关基因表达的影响。方法:分别测定不同浓度芦荟素作用后的Melan-a鼠黑素细胞株的细胞增殖率、酪氨酸酶(TRY)含量及黑素量变化,逆转录-聚合酶链反应(RT-PCR)测定酪氨酸酶相关蛋白(TRP)-1和TRP-2的表达,并与相应浓度的氢醌进行比较。结果:0.01～10.00mmol/L的芦荟素对黑素细胞的增殖率无影响,芦荟素可显著抑制Melan-a鼠黑素细胞株的TRY活性和黑素量生成,抑制作用随芦荟素的浓度升高而增加,0.10mmol/L芦荟素处理的Melan-a鼠黑素细胞株的TRY抑制率及黑素量与0.10μmol/L的氢醌相当(P>0.05);芦荟素还能显著下调TRP-1和TRP-2 mRNA的表达水平(P<0.01),但是TRP-1和TRP-2的变化与TRY及黑素量的变化不平行。结论:芦荟素显著抑制Melan-a鼠黑素细胞株的TRY活性、黑素量和TRP-1/TRP-2 mRNA的表达水平,直接测定黑素细胞TRY浓度和黑素量比检测TRP-1/TRP-2mRNA更能确切地反映芦荟素对黑素细胞的影响。

基于Melan解的水平基床系数分析方法及工程运用

弹性地基法具有概念清晰、过程简单等优点在深基坑设计中广为运用,合理选择土体水平基床系数是弹性地基法的关键因素。以往地基水平基床系数多依靠于经验取值,一些学者也曾经尝试采用mindlin解等一些经典的弹性解来确定地基水平基床系数,但在参数的取值以及边界条件的确定方面均存在问题。根据反对称问题的特点,很好地解决了基坑开挖后的边界条件问题,使之符合弹性半空间理论,同时采用考虑应力状态和应力水平的土体参数,使得参数的选取更加符合实际情况。结合理论分析结果与应力路径试验结果,得到了与模量相关的水平基床系数计算方法。将计算得到的水平基床系数运用到杭州市地铁一号线试验段深基坑开挖工程中,并与实测结果进行比较,验证了计算方法的正确性,为杭州地铁后期设计计算、开挖施工提供了理论依据。该方法与传统弹性地基法相比,水平基床系数的取值有明确的计算方法,与连续介质有限元法相比,参数简单、计算简便、概念明确。

Syn、CgA、melan A和inhibin α在肾上腺皮质腺瘤及嗜铬细胞瘤中的表达及意义

目的分析Syn、CgA、melan A和inhibinα在肾上腺皮质腺瘤及肾上腺嗜铬细胞瘤中的表达情况,探讨这组抗体对肾上腺皮质腺瘤及肾上腺嗜铬细胞瘤的诊断与鉴别诊断的意义。方法采用免疫组化方法检测70例肾上腺皮质腺瘤及57例肾上腺嗜铬细胞瘤中Syn、CgA、melan A和inhibinα的表达情况。结果 Syn、CgA、melan A和inhibinα在肾上腺皮质腺瘤中的阳性率分别为87.1%、8.6%、80%和91.4%,而在肾上腺嗜铬细胞瘤中的阳性率分别为91.2%、100%、0和0。结论 Syn、CgA、melan A和inhibinα联合检测对提高肾上腺皮质腺瘤及肾上腺嗜铬细胞瘤的诊断准确性有显著作用。

基于Melan解的地下连续墙结构分析方法

针对深基坑支护的多支撑地下连续墙结构,考虑分步开挖和支撑对墙体的内力和位移以及支撑反力的影响,提出以弹性地基梁为基础的矩阵位移法。在求解矩阵位移法的过程中,确定土体的水平基床系数成为分析地下连续墙变形和内力的关键。Melan给出半无限大弹性空间内部线性荷载作用下的应力和位移解,在Melan解的基础上,通过对其应力解进行积分处理,并结合平面应变问题的物理方程和几何方程,得到条形荷载作用下的位移解,进而得到水平基床系数。将Melan解的矩阵位移法运用到工程实例中,并将实测结果与计算结果进行比较,验证了其可行性和正确性,可为同类工程结构设计提供理论基础。

山奈素对melan-a黑素细胞株黑素生成的影响

目的:观察山奈素对melan-a黑素细胞株黑素生成的影响。方法:山奈素干预melan-a后,观察对黑素细胞增殖率的影响并测定各组酪氨酸酶活性大小及黑素量。结果:山奈素表现出轻度的促melan-a增殖作用(P<0.01),当山奈素浓度在0.001mmol/L至0.1mmol/L之间时,细胞增殖率和浓度呈指数关系,而浓度高于0.1mmol/L后进入平台期。山奈素在1mmol/L浓度时,促黑素合成的作用与同浓度8-MOP类似(P>0.05)。Fontana-Masson染色后与空白对照比较,经山奈素干预的细胞内,黑素颗粒染成黑色,融合成片,明显多于空白对照。结论:山奈素能够激活melan-a黑素细胞酪氨酸酶的活性,并具有促进黑素细胞增殖、使黑素合成量增加的作用。

风化岩层中考虑拉压模量转换的基坑变形计算

针对风化岩层的不同模量以及与位移有关岩土压力,研究基坑工程岩土体变形计算问题。考虑到风化岩体的拉压不同模量,结合平面应变的物理方程和几何方程,建立平面应变问题的本构方程;基于Melan解,考虑不同模量问题与岩土压力和位移的关系,得到一种能够充分考虑基坑宽度、开挖深度、围护结构埋深以及岩土体应力状态的位移解析解,并将所得的理论计算结果应用于工程实际中,验证该计算方法的可行性和实用性。研究结果表明:随着岩土体拉压模量差值的增大岩土体变形越大,初始位移值与岩土体位移成正比。结果可为基坑工程围护结构的设计计算提供理论依据。

Rescue of the albino phenotype by introducing a functional tyrosinase minigene into Kunming albino mice

AIM： To use the tyrosinase minigene as a visual marker to perform microinjection training and improve the techniques related with transgene to greatly elevate the effidency of gene transfer. METHODS： A mouse tyrosinase minigene, i.e., TyBS, in which the 2.25-kb authentic genomic 5＇ non-coding flanking sequence of mouse tyrosinase was fused to a mouse tyrosinase cDNA, was introduced into the fertilized eggs of outbred Kunming albino mice. RESULTS： Of the 11 animals that developed from the injected eggs, two mice （P1 and #8） exhibited pigmented hair （P1） and eyes （P1 and #8）, as confirmed by PCR analysis for the tyrosinase minigene integrated into the genome. When founder P1 was bred to Kunming male mouse, six progeny out of 11 offspring inherited the transgene and the pigmented-eye phenotype. CONCLUSION： Taken together, these results suggest that this minigene encodes the active tyrosinase protein and that its 5＇ flanking region contains the sequences regulating the expression of mouse tyrosinase gene as expected. We have rescued the albino phenotype by introduction and expression of a functional tyrosinase minigene in the Kunming albino mouse and the transgene can be passed to subsequent generation. These findings also indicate that TyBS can be a useful visual marker gene in the co-transgenic experiments.

考虑应力路径基坑变形计算及支护性能研究

基于半无限大弹性空间在条形荷载作用下应力的Melan解,首先根据基坑开挖过程应力状态的变化,建立基坑开挖问题的平面力学分析模型。利用Duncan-Chang曲线模型中的参数计算方法,推导加载和卸载模量公式,进而结合平面应变问题的物理方程和几何方程,建立平面应变问题的本构方程,得到基坑开挖后土体位移计算方法。然后建立土压力与支护结构位移的正弦和幂函数关系曲线,提出土压力计算方法。最后将理论成果应用于工程实践,将土体位移和土压力的理论值与实测数据进行对比分析。研究结果表明:该计算模型得到的基坑变形位移与实测结果吻合较好,验证了土压力与位移计算方法的合理性,同时由监测数据得到锚索预应力随时间的三阶段变化趋势,以及深层水平位移和坡顶竖向位移的匙形分布特点。

Molecular identification and enzymatic properties of laccase2 from the diamondback moth Plutella xylostella (Lepidoptera:Plutellidae)

Laccase（EC 1.10.3.2）is known to oxidize various aromatic and nonaromatic compounds via a radical-catalyzed reaction,which generally includes two types of laccase,Lac1 and Lac2.Lac1 oxidizes toxic compounds in the diet,and Lac2 is known to play an important role in melanizing the insect exoskeleton.In this study,we cloned and sequenced the cDNA of the diamondback moth,Plutella xylostella Lac2（PxLac2）,from the third instar larvae using polymerase chain reaction（PCR）and rapid amplification of cDNA ends techniques.The results showed that the full-length PxLac2 cDNA was 1 944 bp long and had an open reading frame of 1 794 bp.PxLac2 encoded a protein with 597 amino acids and had a molecular weight of 66.09 kDa.Moreover,we determined the expression levels of PxLac2 in different stages by quantitative PCR（qPCR）.The results indicated that PxLac2 was expressed differently in different stages.We observed the highest expression level in pupae and the lowest expression level in fourth instar larvae.We also investigated the enzymatic properties of laccase,which had optimal activity at pH 3.0 and at 35°C.Under these optimal conditions,laccase had a Michaelis constant（Km）of 0.97 mmol L-1,maximal reaction speed（Vm）of 56.82 U mL-1,and activation energy（Ea）of 17.36 kJ mol-1 to oxidize2,2＇-azino-bis（3-ethylbenzothiazoline-6-sulfonic acid ammonium salt）.Type II copper enhanced laccase activity below 0.8mmol L-1 and reduced enzyme activity above 0.8 mmol L-1 with an IC50 concentration of 1.26 mmol L-1.This study provides insights into the biological function of laccase.

23例鼻腔鼻旁窦恶性黑色素瘤临床病理分析

目的研究鼻腔鼻旁窦恶性黑色素瘤的临床及病理特征。方法采用免疫组化SP方法对23例鼻腔鼻旁窦恶性黑色素瘤的临床及病理进行分析。结果23例鼻腔鼻旁窦恶性黑色素瘤中,男性15例(65.2%),女性8例(34.8%);最大年龄80岁,最小年龄45岁,平均年龄57岁;位于鼻腔19例(82.6%),鼻旁窦4例(17.4%),临床表现全部为鼻肿物;病理组织学类型,小圆细胞型12例(52.2%),上皮样细胞型8例(34.8%),梭形细胞型2例(8.7%),多形性细型胞1例(4.3%);含有黑色素13例(56.5%),无黑色素10例(43.5%):免疫组化染色,Vimentin,23例全部呈阳性反应,阳性率为100%,S-100和HMB45呈阳性反应的都为21例:阳性率都为91.3%,MelanA 19例呈阳性反应,阳性率占82.6%。结论23例鼻腔鼻窦恶性黑色素瘤,临床主要表现为鼻肿物,发病部位大多数在鼻腔,联合应用S-100,HMB45和MelanA弥漫阳性在与其组织学相似的肿瘤鉴别中是诊断鼻腔鼻窦恶性黑色素瘤的重要标准。

移动荷载作用下结构弹塑性安定分析方法及其应用研究

为了研究移动荷载作用下结构的安定性问题,以Melan静力型安定定理为基础,通过构造满足静力平衡条件的最佳残余应力场,并求解移动荷载作用下结构中的真实弹性应力场,发展了一种弹塑性安定性计算方法.该方法摆脱了传统安定性分析方法中的数学规划运算,解决了计算中维数障碍的问题.通过与前人研究结果对比,说明本方法的正确性.基于此方法,研究了列车荷载直接作用下路基的静力安定极限的第一类上限kI和静力安定极限kSD,进行轮载间距L和铁轨间距B对路基安定极限的敏感性分析,发现当轮载间距L和铁轨间距B大于等于10a时,轮载间距L和铁轨间距B对路基安定极限不再产生影响;通过分析材料泊松比v和内摩擦角φ对安定极限的影响,表明安定极限随着路基泊松比的增大略有减小,但随着内摩擦角的增大而有明显增加;并提出了一种基于安定极限荷载包络图的路基安定性评价方法和设计方法,据此可对铁路路基进行设计指导和安全性评价.